CN114015718A - Soybean aflatoxin virus infectious cloning vector and construction method and application thereof - Google Patents
Soybean aflatoxin virus infectious cloning vector and construction method and application thereof Download PDFInfo
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- C12N2750/00011—Details
- C12N2750/12011—Geminiviridae
- C12N2750/12022—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
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Abstract
The invention discloses a soybean leaf curl virus infectious cloning vector, a construction method and application thereof, and particularly relates to a method for constructing two positive repeated soybean leaf curl virus genomes on a binary expression vector pBinPLUS through a gene recombination method on the basis of obtaining a soybean leaf curl virus genome complete sequence, and transforming a recombinant vector into an agrobacterium strain EHA105 through electric shock, wherein the obtained virus vector can successfully infect plants such as soybean, Nicotiana benthamiana and the like. The invention provides a mature system for researching the structure and function of the virus genome and the interaction between the virus and the host.
Description
Technical Field
The invention relates to the field of genetic engineering, in particular to a plant DNA virus-Soybean yellow leaf curl virus (SbYLCV) infectious clone vector construction of geminiviridae.
Background
Geminivirus is a plant single-stranded DNA virus widely existing in the world, the size of the geminivirus is about 18nm multiplied by 30nm, and virus particles are in a double icosahedral structure and have no envelopes. The genome of geminivirus is divided into single-component and double-component, and the size of the genome is generally about 2.5-3.0 Kb.
The soybean yellow leaf curl virus (SbYLCV) belongs to monocomponent geminivirus of geminivirus family, and the genome length is 2782 bp. The virus contains 6 open reading frames, in which the viral strands encode V1 and V2, and the complementary strands encode C1, C2, C3 and C4. The virus seriously threatens the production of leguminous crops, particularly soybean crops, and the infected plants show symptoms of leaf curling, yellowing, growth and development retardation and the like.
The use of disease-resistant varieties and the prevention and control of pests are important measures for preventing and controlling the geminivirus, but the existing resistance resources are few, and the resistance is limited to the prevention and control of a few geminiviruses due to the fact that the geminiviruses are various in types and large in variation. Infectious cloning of viruses is the basis for developing reverse genetics studies of viruses, and has important significance for understanding the functions of virus-encoded proteins, screening resistant varieties, studying plant-virus interactions and virus transmission characteristics. For soybean aflatoxin virus, it is currently unknown which soybean varieties are resistant to the virus, and the functions of the virus-transmission mediator and the encoded protein of the virus are unknown.
Therefore, researching the pathogenicity of the soybean yellow leaf curl virus and screening high-quality antiviral soybean varieties become one of the problems to be solved urgently in the field.
Disclosure of Invention
The invention aims to provide a soybean aflatoxin virus infectious cloning vector and a construction method and application thereof. The invention constructs the infectious clone of the soybean aflatoxin virus on the basis of obtaining the complete sequence of the genome of the soybean aflatoxin virus. Two tandem repeats of the sbYLCV genome sequence are constructed into a binary vector pBinPLUS, transcribed, replicated and infected in a host body after agrobacterium-mediated transformation, so as to be used for researching the function and pathogenicity of the virus.
In order to achieve the purpose, the technical scheme adopted by the invention is as follows:
an infectious cloning vector of soybean aflatoxin, which is pBinPLUS-SbYLCV, is prepared by fusing two tandem repeat soybean aflatoxin SbYLCV nucleotide complete sequences onto the pBinPLUS vector, wherein the soybean aflatoxin SbYLCV nucleotide complete sequence is shown as SEQ ID NO. 9.
The construction method of the infectious cloning vector pBinPLUS-SbYLCV comprises the following steps:
(1) extracting total DNA of plants; extracting total DNA of diseased leaves by adopting a CTAB method; obtaining a soybean yellow leaf curl virus complete sequence;
(2) primers SbYLCV-insert1/SalI-F, SbYLCV-insert1-R, SbYLCV-insert2-F and SbYLCV-insert2/EcoRI-R are designed and shown as SEQ ID NO: 1-4;
using the primer pair SbYLCV-insert1/SalI-F, SbYLCV-insert1-R, an approximately 3kb DNA fragment was obtained, insert1, which was 1 copy of the genome in size;
using the primer pair SbYLCV-insert2-F, SbYLCV-insert2/EcoRI-R an approximately 3kb DNA fragment was obtained, insert2, of size 1 copy of the genome; purifying the amplified PCR product to obtain two SbYLCV nucleotide complete sequences containing different enzyme cutting sites;
(3) carrying out recombination reaction on the purified amplified target fragment and a pBinPLUS vector subjected to double enzyme digestion by SalI and EcoRI, then transforming a ligation product into escherichia coli DH5 alpha, and selecting a positive clone;
(4) extracting and culturing a plasmid containing a target clone, and screening out a clone with two tandem repeat SbYLCV genome nucleotide complete sequences through DNA sequencing to obtain the infectious clone vector pBinPLUS-SbYLCV of the soybean yellow leaf curl virus.
Wherein, in the step (2), PCR amplification is performedThe system is 16 μ L ddH2O, 5. mu.L of 5 XTTrast Fastpfu Buffer, 2. mu.L of 2.5. mu.M dNTPs, 0.4. mu.L of each of upstream and downstream primers (10. mu.M), 1. mu.L of template DNA, and 0.2. mu.L of TransStart Fastpfu DNA Polymerase, wherein the total reaction system is 25. mu.L; the total reaction conditions were: 2min at 95 ℃; 35 cycles of 95 ℃ 20sec, 55 ℃ 20sec, 72 ℃ 3 min; 10min at 72 ℃.
Wherein, In the step (3), the recombination reaction adopts a10 mu L system, wherein 2 mu L of 5 XIn-Fusion HD Enzyme Premix, 2 mu L of SalI and EcoRI double-Enzyme digestion linearization cloning vector pBinPLUS, 0.5 mu L of insert1, 1 mu L of insert2, and ddH is finally used2O adjusted the system to 10. mu.L.
The infectious cloning vector of the soybean yellow leaf curl virus can be used for analyzing the pathogenicity of the soybean yellow leaf curl virus, screening antiviral soybean varieties as a virus source and researching the gene function of the soybean.
The invention also provides an agrobacterium strain carrying the soybean aflatoxin virus infectious clone, and the agrobacterium containing the SbYLCV infectious clone can be obtained by introducing the infectious clone vector into the agrobacterium strain EHA105 by an electric shock transformation method.
The agrobacterium strain can be used for pathogenicity analysis of soybean yellow leaf curl virus and virus gene function research.
Compared with the prior art, the invention has the outstanding effects that:
(1) the agrobacterium strain provided by the invention can produce infectious clone of soybean geminivirus;
(2) the infectious clone provided by the invention can systematically infect soybeans, can efficiently infect solanaceous plant Nicotiana benthamiana, causes obvious virus symptoms, and has simple operation and good repeatability;
(3) the infectious clone provided by the invention can be effectively used for pathogenicity analysis of soybean geminivirus;
(4) the mutant virus gene can be used for researching the function of virus gene.
The construction method and application of the infectious clone vector of soybean aflatoxin virus of the invention are further explained by the following description and specific examples in conjunction with the accompanying drawings.
Drawings
FIG. 1 is a schematic diagram of the construction of the infectious cloning vector pBinPLUS-SbYLCV amplification.
FIG. 2 is the phenotype of pBinPLUS-SbYLCV infectious clones inoculated with Nicotiana benthamiana, tomato;
wherein, A: healthy soybean control inoculated with pBinPLUS empty vector; a: symptoms 14 days after SbYLCV inoculation of soybeans;
b: pBinPLUS empty vector inoculated burley tobacco control; b: symptoms after 30 days of SbYLCV inoculation with burley tobacco.
FIG. 3 shows the PCR detection results of pBINPLUS-SbYLCV inoculated with soybean and Nicotiana benthamiana. Wherein, M: DNA Marker; 1. 2: healthy Bungarus benthamii control inoculated with pBinPLUS empty vector; 3. 4: SbYLCV inoculated burley tobacco; 6. 7: healthy soybean control inoculated with pBinPLUS empty vector; 8. 9: SbYLCV inoculated soybeans; 5. 10: negative control for PCR.
Detailed Description
The examples provided by the present invention are, without specific reference, based on routine experimental conditions, wherein the primer sequences used are shown in Table 1.
TABLE 1 SbYLCV infectious cloning vector construction primers
Example 1 cloning and determination of the complete sequence of the genome of the Soybean Aflatopsis flava Virus
(1) Collection of Soybean sample suspected of being infected with geminivirus
(2) Extraction of plant Total DNA
The CTAB method is adopted to extract the total DNA of the diseased leaves, and the specific operation is as follows: 0.1g of fresh plant leaves are weighed into a tube and after quick freezing with liquid nitrogen ground for 1 minute in a sample grinder (frequency set at 40Hz) to a powder. Adding 700 muL65 ℃ preheated CTAB extract (2% v/v beta-mercaptoethanol was added at the time of use), heated at 65 ℃ for 1h, shaken every 10-20 minutes. Equal volume of 24: 1 chloroform: isoamyl alcohol, mixing and extracting up and down, centrifuging at 4 ℃ and 10000rpm for 5 min. The supernatant was carefully transferred to a new 1.5mL centrifuge tube, 1/10 volumes of 3M NaAc and an equal volume of-20 ℃ pre-cooled absolute ethanol were added, and the mixture was placed in a-20 ℃ refrigerator for 1 h. Centrifuge at 12000rpm for 15min at 4 ℃ and discard the supernatant. The pellet was washed with 1mL of 70% ethanol, centrifuged at 12000rpm for 5min at 4 ℃ and repeated three times. Discarding the supernatant, and oven drying at 37 deg.C for 10-15 min. After drying, 50. mu.L of ddH was added2And dissolving the O. Adding 1 μ L RNAase (10 μ g/. mu.L), mixing, and digesting at 37 deg.C for 30 min. Storing at-20 deg.C.
(3) SbYLCV full sequence amplification and genomic sequence determination
Taking about 100ng of sample DNA, amplifying a DNA fragment of about 3.0kb by using a pair of back-to-back primers, cutting and recovering the gel, connecting the gel to a whole-scale gold pEASY-Blunt Simple Cloning T vector, transforming the recombinant vector into escherichia coli DH5 alpha, and screening out positive clones by using universal primers M13F and M13R; the positive clones were selected and cultured overnight, and plasmids containing the target clones were extracted and verified by DNA sequencing. The obtained sequence is shown as SEQ ID NO. 9; the back-to-back primers are shown as SEQ ID NO. 7-8; M13-F and M13-R are shown as SEQ ID NO: 5-6.
EXAMPLE 2 construction of the soybean flaviviruses infectious clone pBinPLUS-SbYLCV (see FIG. 1)
Based on the complete sequence obtained in example 1, primers SbYLCV-insert1/SalI-F, SbYLCV-insert1-R, SbYLCV-insert2-F, SbYLCV-insert2/EcoRI-R were designed, using the primer pairs SbYLCV-insert1/SalI-F, SbYLCV-insert1-R, to obtain a DNA fragment of about 3kb, i.e.insert fragment 1, of size 1 copy of the genome; using the primer pair SbYLCV-insert2-F, SbYLCV-insert2/EcoRI-R an approximately 3kb DNA fragment was obtained, insert2, of size 1 copy of the genome; and purifying the amplified PCR product to obtain two SbYLCV nucleotide complete sequences containing different enzyme cutting sites.
Wherein, the PCR amplification system is 16 muL ddH2O, 5. mu.L of 5 XTStart Fastpfu Buffer, 2. mu.L of 2.5. mu.M dNTPs, upstream and downstreamPrimers (10. mu.M) were 0.4. mu.L each, 1. mu.L of template DNA, and 0.2. mu.L of TransStart FastPfu DNA Polymerase, and the total reaction system was 25. mu.L. The total reaction conditions were: 2min at 95 ℃; 35 cycles of 95 ℃ 20sec, 55 ℃ 20sec, 72 ℃ 3 min; 10min at 72 ℃.
Carrying out recombination reaction on the purified amplified target fragment and a pBinPLUS vector subjected to double enzyme digestion by SalI and EcoRI, transferring a ligation product into escherichia coli, and selecting a positive clone; extracting and culturing a plasmid containing a target clone, and screening out a clone with two tandem repeat SbYLCV genome nucleotide complete sequences through DNA sequencing to obtain the infectious clone vector pBinPLUS-SbYLCV of the soybean yellow leaf curl virus.
Wherein the recombination reaction employs a 10. mu.L system, wherein 2. mu.L of 5 XIn-Fusion HD Enzyme Premix, 2. mu.L of SalI and EcoRI double-digested linearized cloning vector pBinPLUS (100ng), 0.5. mu.L of insert 1(100ng), 1. mu.L of insert 2(130ng), and finally ddH is used2O adjusted the system to 10. mu.L.
Example 3 pBinPLUS-SbYLCV infectious clone inoculation
The pBinPLUS-SbYLCV is transformed by electric shock and introduced into agrobacterium strain EHA105, after colony PCR verification, single spots are picked and inoculated into YEP culture medium containing kanamycin (50mg/L) and rifampicin (50mg/L) resistance for overnight shaking culture at 28 ℃, and when the OD of the cultured bacterial liquid is600Centrifuging at 4000rpm for 10min at 1.0-1.5, discarding supernatant, collecting thallus, and inoculating with inoculation buffer (containing 10mM MgCl2ddH of 10mM MES (pH5.6) and 100mM acetosyringone2O solution) and adjusting the bacterial liquid concentration to OD600Standing at room temperature in dark place for 2h about 1.0. Selecting Benshi tobacco of 4-5 leaf stage, slightly pricking several small holes on the back of plant leaf blade during injection, and slowly inoculating bacterial liquid into leaf blade tissue through the small holes. 8-10 plants were injected for each treatment and the plants were inoculated and placed in a greenhouse at 25 deg.C (16 hours light/8 hours dark alternating).
EXAMPLE 4 infectious assay of pBinPLUS-SbYLCV infectious clones
The plants were observed for symptoms at regular intervals and the changes induced on the different plants were monitored. PCR detection was performed on plants with or without symptoms. Symptom observation shows that systemic infected leaves after agrobacterium infiltration and inoculation of the Nicotiana benthamiana have obvious symptoms of new leaf rolling and leaf shrinkage, curling and malformation (as shown in figure 2); systemic leaves showed marked leaf rolling, shrinkage and floral leaf symptoms after soybean inoculation (see figure 2).
PCR detection using virus specific primers, the B.benthamiana and soybean samples inoculated with pBinPLUS-SbYLCV were amplified to a specific band of about 1700bp (see FIG. 3). These results show that the pBinPLUS-SbYLCV infectious clone constructed has biological activity and can successfully infect Nicotiana benthamiana and soybean.
The above results show that: the pBinPLUS-SbYLCV infectious clone constructed by the invention has biological activity and can successfully infect the Nicotiana benthamiana and soybean.
The above-mentioned embodiments are merely illustrative of the preferred embodiments of the present invention, and do not limit the scope of the present invention, and various modifications and improvements of the technical solution of the present invention by those skilled in the art should fall within the protection scope defined by the claims of the present invention without departing from the spirit of the present invention.
Sequence listing
<110> institute of plant protection of Chinese academy of agricultural sciences
<120> soybean aflatoxin virus infectious cloning vector, and construction method and application thereof
<130> DS211-055
<160> 9
<170> SIPOSequenceListing 1.0
<210> 1
<211> 41
<212> DNA
<213> SYLCV-insert1/SalI-F(Artificial Sequence)
<400> 1
cttgcatgcc tgcaggtcga ccattcctca agttcttccg g 41
<210> 2
<211> 30
<212> DNA
<213> SYLCV-insert1-R(Artificial Sequence)
<400> 2
ttgaggaatg gatgtcagag aacgtcaagg 30
<210> 3
<211> 30
<212> DNA
<213> SYLCV-insert2-F(Artificial Sequence)
<400> 3
ctctgacatc cattcctcaa gttcttccgg 30
<210> 4
<211> 41
<212> DNA
<213> SYLCV-insert2/EcoRI-R(Artificial Sequence)
<400> 4
gtaattgtta attaagaatt cgatgtcaga gaacgtcaag g 41
<210> 5
<211> 17
<212> DNA
<213> M13-F(Artificial Sequence)
<400> 5
gtaaaacgac ggccagt 17
<210> 6
<211> 17
<212> DNA
<213> M13-R(Artificial Sequence)
<400> 6
caggaaacag ctatgac 17
<210> 7
<211> 20
<212> DNA
<213> SYLCV-F(Artificial Sequence)
<400> 7
cattcctcaa gttcttccgg 20
<210> 8
<211> 22
<212> DNA
<213> SYLCV-R(Artificial Sequence)
<400> 8
gaatggatgt cagagaacgt ca 22
<210> 9
<211> 2782
<212> DNA
<213> Soybean aflatoxin Virus SYLCV (Artificial sequence)
<400> 9
accggatggc cgcgaaaaaa tttagggcac gtgtccaaac ttagtggcca agctattagt 60
gcgatgtgga ccccccccgg ggcgcttata ggctcccttg ttttgtccta tataagtttt 120
tactgtcgtc ttttctttat ggaggaccga aatattgtcg tccactttac ctttgatccg 180
caacacccac gtagaatttt gaaatctctt agtgggtttg tgattagccg ttgtagtgcg 240
gagcagttaa tccgttggat taatggatta cagcaggaag agaaagaatt attcagggcc 300
ctcaacgcct gttccgggga cacccaagag aggcaggcgc ggttacggtc aatcatggaa 360
cccagctgca aggcggaggt taaacttcgg tgcaatgaac cgctccataa ttccgaagcc 420
gataaacagg acgaatcttc aggtgaatga tgtatctatt ggtggtgacc agtcctcttc 480
atttagtatt atcaatggat ctgggcttgt ctatttacta aatgggtatg agttgggcaa 540
tgggaacgct catcgtcatt cgaataagac ttacacctat aagaccatga ttaggattgc 600
gcttatgggt gactctgatt attatgcgtc gtgttattcc cagacgttct tttggtggtt 660
aatttatgat cagagctcct ttgctgatag acctggggct aatcagatat tggatctgac 720
caagggtccg gaggctgcta ccttgttaag ggatcagtct gacaggttca aagtccatgc 780
aaagggacgt aagaggctct attctaattc cattatacag tcagctaaat caagttacat 840
agccggatca cctcctacgt ttccatacat ggagggtatg tttcggactc cgtctgcaac 900
tgaattcaag gatgaagtaa ctaccggggg ttatggggat tataagaagg ggtgtttgta 960
ttttgtaatt gttcctgaca ttgcttggac taatgtaaaa agtggaatac ggggcaaaat 1020
acgcacgtat ttcaagagca gattgtaatg aaatgaaata cttaatataa attgcattgt 1080
cttaatataa attgcattga atgtttaaat tgtctacata cattcgatct atccaaacac 1140
acttgtctat tacataatgc aaagcatcaa ttacatcctt aattgaaata actcctaaat 1200
tattaagata atacataata tgaaacctaa ttctagatag aacggatgtc ggaccagcag 1260
tcgtcaaggt cgtccagacc ttcaaatgca gccaacattt gtgtagtccc aaggctttgc 1320
gaaggttgtg gttgaacatg actcgtaact ggaaggcttg tcgatatgga ggagtggttg 1380
tcgcccaatc cctctcgagt ttgaacgaga ttgggttctt taagctgtcg taatatacgc 1440
tggaaatcat ttggatgtgg gtaattgatt cccccgtgcg ataatccgtg agggcattca 1500
ggatggacgt agaagaggca taagccacaa caggcattta ttctgcgcct tctaggtggt 1560
ttcttcttgg cttgcttgtg aataattttc tgggggagga gtgctttgga tattgtacaa 1620
ggggttcgtg atggtgacga agattgcatt tttcaatgcc cactgtttca attcccgatt 1680
cttatcttca tctaaaaact ctttatacga agaattgggt cctggattgc agaggaagat 1740
agtgggtata ccacctttaa tttgaatggg cttgccgtat tttgtgttgc tttgccagtc 1800
cctttgggcc cccataaatt ctttaaagtg ctttagataa tgcgggtcaa cgtcatcaat 1860
gacgttatac caggcatcat tactgtacac tttgggacta agatcaagat gaccacataa 1920
ataattatgt ggacccaatg aacgagccca catagttttt cctgttctac tttcaccttc 1980
aacaactata ctcatgggtc tccatggccg cgcagcggca tccttgacgt tctctgacat 2040
ccattcctca agttcttccg gaacttgatt gaaagaagaa gaagaaaaag ggcaaacaaa 2100
tacttctaaa ggagctgcga aaatcctatc tagattagaa tttaaattat gatactgtaa 2160
tataaaatct ttaggcatta attccctaat caaattaaga gcctctgact tactgccgct 2220
gttaagcgcc ttggcgtaag catcgttggc tgattgttgt cctcctcgtg cagatcgtgc 2280
gtcgatctga aactgtcccc attcgagggt gtctccgtcc ttgtcgatat aggacttgac 2340
gtcggagctg gatttagctc cctgtatatt tggatggaaa tatgctgacc tggttgggga 2400
tagcaggtcg aagaatctgt tattcgtgca gacgagtttc ccttcgaatt gcataagcac 2460
atggagatga gggctcccat cttcgtgaag ttctctgcag attttgatat agagcttatt 2520
gacagcagta tctaatttct gtaattggtc tagagtttcc tctttggtta gagaacaatg 2580
gggatatgta aggaaatagt ttttggcttg tatcctaaaa cgacgtggag gagccatttt 2640
atgtgtcgtt ttggattgga gacattcact aactcgtatg gcattggaga caggagtaca 2700
atatataggt gtctctaaat ggcattattg taatttggta aaggtagagt tgaaatttga 2760
aagcggccat ccgtataata tt 2782
Claims (7)
1. An infectious cloning vector of soybean aflatoxin virus, which is characterized in that: the infectious cloning vector is pBinPLUS-SbYLCV, and is prepared by fusing two tandem repeat soybean yellow leaf curl virus SbYLCV nucleotide complete sequences to the pBinPLUS vector, wherein the soybean yellow leaf curl virus SbYLCV nucleotide complete sequence is shown as SEQ ID NO. 9.
2. The method for constructing the infectious cloning vector of soybean aflatoxin virus according to claim 1, which comprises the following steps:
(1) obtaining a complete sequence of soybean aflatoxin virus SbYLCV nucleotide;
(2) primers SbYLCV-insert1/SalI-F, SbYLCV-insert1-R, SbYLCV-insert2-F and SbYLCV-insert2/EcoRI-R are designed and shown as SEQ ID NO: 1-4;
using the primer pair SbYLCV-insert1/SalI-F, SbYLCV-insert1-R, an approximately 3kb DNA fragment was obtained, insert1, which was 1 copy of the genome in size;
using the primer pair SbYLCV-insert2-F, SbYLCV-insert2/EcoRI-R an approximately 3kb DNA fragment was obtained, insert2, of size 1 copy of the genome; purifying the amplified PCR product to obtain two SbYLCV nucleotide complete sequences containing different enzyme cutting sites;
(3) carrying out recombination reaction on the purified amplified target fragment and a pBinPLUS vector subjected to double enzyme digestion by SalI and EcoRI, then transforming a ligation product into escherichia coli DH5 alpha, and selecting a positive clone;
(4) extracting and culturing a plasmid containing a target clone, and screening out a clone with two tandem repeat SbYLCV genome nucleotide complete sequences through DNA sequencing to obtain the infectious clone vector pBinPLUS-SbYLCV of the soybean yellow leaf curl virus.
3. The method for constructing an infectious cloning vector of soybean aflatoxin virus according to claim 2, which is characterized in that: the PCR amplification system in the step (2) is 16 mu L ddH2O, 5. mu.L of 5 XTStart FastPfu Buffer, 2. mu.L of 2.5. mu.M dNTPs, 10. mu.M of each of the upstream and downstream primers, 0.4. mu.L of template DNA, 0.2. mu.L of TransStart FastPfu DNA Polymerase, and 25. mu.L of total reaction system; the total reaction conditions were: 2min at 95 ℃; 35 cycles of 95 ℃ 20sec, 55 ℃ 20sec, 72 ℃ 3 min; 10min at 72 ℃.
4. The method for constructing an infectious cloning vector of soybean aflatoxin virus according to claim 2, which is characterized in that: the recombination reaction In step (3) adopts a 10. mu.L system, wherein 2. mu.L of 5 XIn-Fusion HD Enzyme Premix, 2. mu.L of SalI and EcoRI double Enzyme tangent lineSex cloning vector pBinPLUS, 0.5. mu.L insert1, 1. mu.L insert2, finally with ddH2O adjusted the system to 10. mu.L.
5. The infectious cloning vector of soybean aflatoxin virus of claim 1 for use in molecular biology for analyzing pathogenicity of soybean aflatoxin virus, screening antiviral soybean varieties as a source of poison, and studying soybean gene function.
6. An agrobacterium strain carrying soybean aflatoxin infectious clone is characterized in that: agrobacterium containing the SbYLCV infectious clone is obtained by introducing the infectious cloning vector of claim 1 into Agrobacterium strain EHA105 by electroporation.
7. The use of the agrobacterium strain of claim 6 in pathogenicity analysis of soybean aflatoxin viruses, viral gene function and soybean gene function research.
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| CN114807216A (en) * | 2022-04-30 | 2022-07-29 | 南京农业大学 | Soybean disease green related virus infectious cloning vector and soybean high-efficiency inoculation method |
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| CN102428187A (en) * | 2009-04-20 | 2012-04-25 | 孟山都技术公司 | Multiple Virus Resistance In Plants |
| CN113403335A (en) * | 2021-06-22 | 2021-09-17 | 中国农业科学院植物保护研究所 | Mulberry leaf type dwarf associated virus MMDaV infectious clone vector and construction method and application thereof |
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| CN114807216A (en) * | 2022-04-30 | 2022-07-29 | 南京农业大学 | Soybean disease green related virus infectious cloning vector and soybean high-efficiency inoculation method |
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